Immunoblot of cell cycle‐associated proteins expressed in MDM. Star indicates non‐specific band. This Western blot quantification is from Donor 1 in Fig 2A. The same blots were used in Fig 2A to allow comparison of different cell cycle‐associated proteins, as well as SAMHD1.

Uninfected MDM or MDM exposed for 48 h to VSV‐G HIV‐1 GFP (HIV‐1 exposed) were stained for cell cycle‐associated proteins MCM2, Geminin and EdU incorporation (EdU added to MDM 50 h prior to analysis). On average, 104 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ. Diagram of the cell cycle pathway with associated markers is also shown.

Cell cycle analysis by quantitation of DNA content by flow cytometry. Cycling THP‐1 cells and both unstimulated and stimulated MDM were labelled by propidium iodide (PI) and analysed by FACS.

CFSE loaded MDM were cultured for 4 days to determine cell division/proliferation by FACS.

The titres of released viruses were determined by infection of the indicator cells HeLa TZM‐bl. Culture supernatants from BaL‐infected MDM were harvest at day 2, 6 and 9 post‐infection, filtered and used to infect TZM‐bl cells. Luciferase activity of the TZM‐bl cells was measured 24 h post‐infection.

MDM were infected with equal amounts of p24 (50 ng) of BlaM‐Vpr‐containing viruses for 4 h. Cells were loaded with CCF2/AM dye, and fusion events were detected by flow cytometry using BD LSR Fortessa and gated from 10,000 cells.

MDM differentiated and cultured in RPMI complemented with MCSF and 10% human serum for 7 days were changed into stimulatory medium (10% FCS) and cultured for additional 3 days. Cells were infected with VSV‐G‐pseudotyped HIV‐1 GFP and the percentage of infected cells detected 48 h post‐infection by FACS.

MDM were treated with the CDK4/6 inhibitor Palbociclib (1 μM) 18 h before infection and infected with VSV‐G‐pseudotyped GFP virus; VLP‐vpx was added at the time of infection. Cells from this experiment were lysed and used for immunoblotting.

A. MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1.

C. MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection (n = 2, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, unpaired t‐test).

D. Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days.

E. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m.

G–I MDM were infected with VSV‐G‐pseudotyped HIV‐1 GFP in the presence or absence of VLP‐vpx, stained and recorded 48 h post‐infection. On average, 104 cells in each experiment were recorded and analysed for infection (G), MCM2 expression (H) and co‐localisation between infection and MCM2 protein (I) using Hermes WiScan cell‐imaging system and ImageJ (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, ***P‐value ≤ 0.001, unpaired t‐test).

J. Log odds ratios calculated from quantifications of stimulated MDM show association of infection with MCM2/EdU (1, high association; 0, no association) (n ≥ 3, mean ± s.e.m.). Immunoblot shows expressions levels of SAMHD1 in these experiments. EdU was added to cells at the time of infection.

Figure EV4.MDM in a G1‐like phase are highly permissive to HIV‐1 infection

A. MDM were cultured in the presence of 5 μM EdU for 72 h and labelled using Click‐iT® EdU Alexa Fluor® 488 Imaging Kit. The percentage of EdU‐positive cells was detected by using Hermes WiScan and ImageJ.

B, C MDM were infected with VSV‐G‐pseudotyped HIV‐1 GFP or Semliki Forest virus (SFV) stained and recorded 48 h post‐infection and analysed for co‐localisation between infection and MCM2 protein or EdU incorporation (active DNA synthesis, EdU was added to cells at the time of infection). Co‐localisation analysis between infection and MCM2 in the presence or absence of SAMHD1 protein is also shown.

C. Graph shows the percentage of MCM2‐positive cells from four independent experiments. On average, 2 × 103 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ (mean ± s.e.m.).

D. Stimulated MDM were treated with 1 μM SAHA, MDM were lysed, and immunoblotting was performed to detect cell cycle‐associated proteins.

E, F MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were (E) lysed and used for immunoblotting or (F) inspected for infection. Scale bars: 20 μm. On average, 104 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ (n ≥ 2, mean ± s.e.m.).

MDM were treated with panobinostat (PANO) 18 h before infection with VSV‐G‐pseudotyped HIV‐1 GFP. The percentage of infected cells was detected by FACS 48 h post‐infection.

Quantification of specific protein band intensities from the immunoblot in Fig 5D using a CCD camera. Intensities of proteins bands were normalised to intensity of the actin protein band.

MDM were treated with 1 μM SAHA (Vorinostat), and then recorded and analysed for infection (VSV‐G HIV‐GFP) using Hermes WiScan. Scale bars: 20 μm.

MDM were treated with 1 μM SAHA (Vorinostat) and co‐infected with VSV‐G‐pseudotyped HIV‐1 GFP and VLP‐vpx. Cells from a representative donor were lysed and used for immunoblotting. The percentage of infected cells was detected by Hermes WiScan.

MDM were treated with 0.025 μM panobinostat and then recorded and analysed for infection (VSV‐G HIV‐GFP) using Hermes WiScan. Scale bars: 20 μm.

MDM were treated with 0.025 μM panobinostat and co‐infected with VSV‐G‐pseudotyped HIV‐1 GFP and VLP‐vpx. Cells from a representative donor were lysed and used for immunoblotting. The percentage of infected cells was detected by Hermes WiScan.

Data information: On average, 104 cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system and ImageJ (n ≥ 3, mean ± s.e.m.). Source data are available online for this figure.